The three-component methane monooxygenase (MMO) catalyzes the first step of CH4 oxidation to CO2 in methanotrophs. In many ecosystems, this reaction is the primary barrier limiting the methanogens. Formation of a powerful oxidating intermediate by oxygen activation at the 2Fe center of the MMO hydroxylase and substrate oxidizing intermediate by oxygen activation at the 2Fe center of the MMO hydroxylase and substrate oxidation are regulated by binding of two other MMO protein components, namely the "effector" component, MMOB and the reductase, MMOR. This proposal uses site-directed spin and fluorescent labeling coupled with nitroxide-EPR and fluorescence energy transfer (FET), respectively, to study the complexes between the MMO components. The results will be used to correlate structural changes in wild type and mutant MMOBs with the dramatic kinetic effects observed in previous and current studies and to determine the role of the topography of the MMOH-MMOB-MMOR ternary complex in the regulation of electron transfer and catalysis.